Immunoassay assembly and methods of use

ABSTRACT

The present invention relates to an improved system for efficiently and accurately performing immunoassays, such as ELISAs. The invention provides an immunoassay assembly which includes a flow-through unit and an aspiration pump. The immunoassay flow-through unit includes an outer seal; at least one bed support; an inner seal; and a packed non-porous bed. The unit is releasably attached to an aspiration pump which enables the controlled flow rate of liquid passing through the packed bed of the flow-through unit. The invention also provides a method of using the immunoassay assembly to identify analytical targets of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional patentapplication Ser. No. 60/590,673 filed Jul. 23, 2004.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

None.

BACKGROUND OF THE INVENTION

The fields of life science research and pharmaceutical development arecritically dependent upon highly selective and sensitive quantitativeassays for a wide range of different biomolecules (such as proteins,antibodies, cytokines, receptors, enzymes, peptides, nucleic acids,hormones, and the like) in complex clinical or biological samples (suchas blood, urine, tissue or cellular extracts, cell culture supernatants,bioprocess feedstreams, and the like). In typical samples (which maycontain thousands of different molecular species) the analytes ofinterest may be present at extremely low concentrations (nanograms permilliliter or less), but the samples may be available only in very smallquantities (microliters or less). The rapid growth in the field ofbiotechnology and the introduction of many potential new drug targetsfrom genomic research have created an increasing demand for more rapidand efficient analytical methods, without any sacrifice in performance.

In order to simultaneously obtain high selectivity (the ability tomeasure one very specific molecule in a complex mixture) and highsensitivity (the ability to accurately quantify very smallconcentrations or amounts), a number of analytical methods have beendeveloped which couple powerful molecular separations with extremelyresponsive detection methods.

One of the most widely used of these separation-based methods is theEnzyme-Linked Immuno-Sorbent Assay or ELISA. In ELISA, an antibody isimmobilized on a solid phase support and exposed to a liquid sample,enabling any antigen (analytical target) to bind specifically to theantibody. Non-binding molecules in the sample are washed away. The solidphase with bound target can then be exposed to either antigen or asecond antibody specific to the target that are labeled with a linkedenzyme. After the non-binding labeled molecules are washed away, thesolid phase is then exposed to enzyme substrate under controlledconditions so that the amount of colored or fluorescent enzyme productformed is proportional to the amount of label present, which can be usedin turn to quantify the amount of target present in the original sample.

Currently in the fields of life science research and pharmaceuticaldevelopment, ELISAs are done almost entirely using plastic (typicallypolystyrene) multi-well plates called microtiter plates or microplates.The wall of each well serves as both the solid phase for binding theantibody and antigen, as well as the container for the sample andreagents that are added. Liquid addition is done by pipetting, andwashing is done by rapidly pipetting a wash solution in and out of thewell. Readout of the enzyme product is done through the transparentplastic wells with an optical plate reader that measures eitherabsorbance or fluorescence. This technique is quite simple, requiresminimal specialized equipment and is very flexible in terms of thereagent systems and assay formats that can be used.

However, the microplate ELISA method suffers from a number of seriousdrawbacks. The most important is that the antibody is bound to the wallof the well, and thus the only way sample and reagent molecules canreach the surface to interact is by molecular diffusion. Diffusion is arelatively slow process over the potential path length of severalmillimeters found in a typical microplate well, and so after liquids areadded for each step, the user must allow the plate to incubate foranywhere from 30 minutes to several hours to overnight to allow thebinding reaction to approach equilibrium. This makes the total assayturnaround time quite long, typically on the order of 4 to 24 hours.

In addition, microplate ELISAs are subject to a high degree ofvariability, due to the critical techniques required. The pipetting mustbe done very accurately and consistently into each well, and timingbetween wells can be very important. Temperature variation between theinner and outer wells in a plate can lead to variability, as can jarringor vibration of the plates during incubation. Most operators are not ascareful as required due to the tedium of the work, and assaycoefficients of variation of 10 to 30% or more are not uncommon.Automation of microplate ELISAs using conventional liquid handlingrobotic equipment is possible, but is quite complex and often does notimprove reproducibility. Users often find that such automated assaysmust be constantly monitored by a human operator to prevent problems.

A related set of highly selective separations are used in amicro-preparative mode to isolate the target from a complex sample inpreparation for mass spectroscopy (MS), using either an ElectroSprayInterface (ESI) or Matrix Assisted Laser Desorption Interface (MALDI) toionize the sample upon entry into the instrument. MS is unique in itsability to very rapidly provide comprehensive identity and structuralinformation on analyte molecules with high sensitivity from very smallvolumes of sample. Because of the rich structural information MS givesabout individual molecular species (especially proteins), complexsamples must be fractionated or at least significantly simplified toenable a meaningful MS analysis to be performed. Purification methodsare also needed when the target of interest is present in very smallconcentrations relative to other components in the sample, as is oftenthe case in clinical or biological samples. Once the samples areseparated into individual fractions or peaks, additional processing(such as concentration, desalting, enzymatic digestion and/or matrixaddition) often must be performed to prepare the sample for analysis bythe MS instrument.

In sample prep for MS, the target molecules are selectively bound to asurface by immobilized antibodies or other selective surface groups(such as ion exchange, reversed phase, hydrophobic interaction,affinity, and the like), and non-binding contaminants are washed away.Then the bound target is eluted (using for example salt, acid or organicsolvent) for collection into a tube or on a surface spot for furtheranalytical processing. It is also possible to immobilize an enzyme (suchas a protease or glycosidase) to the packed bed to enable very rapidprocessing of the target molecule prior to further analysis. The amountsof target analyte required for MS are very similar to those required fordetection using an ELISA.

Currently two separation methods are most often used as a front-end forMS and for two-dimensional gel electrophoresis and for gradient highperformance liquid chromatography (HPLC). Both of these techniques arepowerful and work reasonably well for comprehensively searching throughall of the components in complex samples. However, these methods are notwithout problems. Two-dimensional gels, for example are labor-intensive,have many steps, and require many hours or even days to complete(compared to the analysis time of the MS, which is usually a matter ofseconds). HPLC is sometimes not compatible with large proteins, andinstrumentation systems with comparable throughput can be almost asexpensive and complex as the MS itself. Sample carryover can also be anissue in high throughput applications.

Many different types of small-scale adsorption-based separation deviceshave been developed, and some are offered for use in MS samplepreparation. Most have been adapted from devices designed for solidphase extraction (SPE) used in general analytical chemistry. One popularapproach is the “spin column”, in which a small packed bed is suspendedin a microcentrifuge tube, with samples and eluents driven through usinga laboratory centrifuge. Some spin columns are also designed to bedriven by a vacuum manifold. Spin columns are offered by a number ofvendors in a range of common surface chemistries (reversed phase, ionexchange, metal chelate affinity). Although they are simple, spincolumns suffer from the need to collect the final product in a testtube, then transfer it by pipette to the next step in the process or tothe MS interface. These sample transfer steps can lead to significantlosses, especially with dilute samples. Spin columns are poorly suitedfor automation. Also, most of the available spin columns are too large(typical bed volumes of 10 to 200 μL) for handling sample volumes in thelow microliter range or below. It is also virtually impossible tocontrol the flow rate through a spin column with any precision, whichcan reduce capture efficiency and reproducibility.

Perhaps the most popular approach to simplified sample preparation forMS is the use of modified pipette tips containing adsorbent materials.In the Millipore ZipTip product, a standard chromatographic adsorbent isembedded in a sponge-like polymer matrix in the end of the tip. Thematrix enables flow by aspiration in a standard pipettor with littlepressure drop. The company has also made this technology available in a96-well plate format (ZipPlate) driven by a vacuum manifold, primarilyfor use in in-gel digestion and purification of 2D gel spots. Glygen hasdeveloped a tip with a flattened area at the end with the adsorbentparticles embedded thermally on the inner surface, which can handlesample volumes as low as 1 to 10 μL. PhyNexus produces pipette tipscontaining affinity chromatography resins sandwiched between sealed-onscreens in standard 200 and 1000 μL pipette tips. The tips produce finalproduct in an elution volume of 10 to 15 μL. These pipette tip productsare simple and convenient, but suffer from a number of drawbacks. Ifused with syringes or pipettors, it is very difficult to achievesufficiently slow flow rates for complete binding, especially whenaffinity or antibody separations are used. As a result, multipleaspirate/dispense cycles are needed. This, in turn, leads tonon-quantitative and/or non-reproducible capture of the bound targetproviding typical recoveries for proteins only in the 20 to 40% range.Like spin columns, pipette tips can only perform one separation step ata time, with some type of transfer operation required between steps,with likely concomitant sample loss. Flow through the pipette tip canonly go in and out through the distal port, which limits the flexibilityof operation.

A number of academic labs and companies have worked to integrate theseparation and other processing steps or improve MS sensitivity throughmodifications to the MALDI plate itself. One example is the SELDI(Surface-Enhanced Laser Desorption Ionization) ProteinChip product fromCiphergen Biosystems. In this approach, various surface chemistries areincorporated into a spot on the plate to effect physical adsorption, ionexchange, or separations with affinity binding using antibodies orreceptors. etc.). A small volume of sample is incubated on the spot, thenon-binding materials washed off, and then matrix is added prior toanalysis. The MALDI plate approaches are, of course, not amenable foruse in electrospray MS. They are also limited to use with a singlebinding selectivity, so that other separation and preparation steps mustbe carried out elsewhere. The amount of sample that can be processed inthis manner is also limited, so significant concentration is difficultto achieve.

A combined system approach has been developed by Intrinsic Bioprobes.The Mass Spectrometric ImmunoAssay (MSIA) technology developed by thiscompany uses pipette tips incorporating a porous glass frit, onto whichantibodies are immobilized. The bound antigens isolated from samples areeluted onto a MALDI plate for analyis. In other products, a pipette tipantibody-based separation device (using a porous glass monolith solidphase) is used in combination with enzymes (such as trypsin) immobilizedon the MALDI plate. Gyros AB has developed a microfluidic system in theform of a compact disk (CD)—shaped device that incorporates severalseparation steps (including antibody affinity) driven by centrifugalforce. The major application for this system are ELISA and samplepreparation prior to MALDI MS. Bruker Daltonics has introduced theClinProt system for purification prior to MALDI MS based upon roboticliquid handling and magnetic beads. Other integrated systems have someinteresting advantages, but most of them require complex and expensivededicated instrumentation for implementation.

Thus the field of biomolecule separation is one in which there is stillroom for improvement to overcome some of the limitations in prior artapproaches and standard equipment. In particular, the use of themicrotiter plate is less appropriate today given the sensitivity andspeed desired by modern analytical biochemistry.

BRIEF SUMMARY OF THE INVENTION

The present invention is summarized as a novel system for efficientlyand accurately performing immunoassays, such as ELISAs. One aspect ofthe invention provides an immunoassay assembly including a flow-throughunit and an aspiration pump.

In another aspect, the invention provides a flow-through unit having aninner seal; a pair of bed supports; and a packed particle bed.

In another aspect, the flow-through unit of the invention is releasablyattached to a liquid handling device.

In yet another aspect the invention also provides a method of using theimmunoassay assembly to identify an analytical target, by loading asample solution and a reagent onto a packed bed of the flow-throughunit; aspirating unbound antigen and reagents such as enzyme conjugatesthrough the unit; and identifying the analytical target of interest.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although suitable methods andmaterials for the practice or testing of the present invention aredescribed below, other methods and materials similar or equivalent tothose described herein, which are well known in the art, can also beused. Other objects, advantages and features of the present inventionwill become apparent from the following specification taken inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a cross-sectional view though a flow-through immunoassay unitconstructed according to an embodiment of the present invention.

FIG. 2 is a cross-sectional view of the assay unit of FIG. 1 with astandard 200 μl pipette tip inserted therein.

FIG. 3 is a cross-sectional view similar to FIG. 2 with a standard 10 μlpipette tip inserted into the assay unit.

FIG. 4 is a cross-sectional view of an assay unit with a standard 20gauge hypodermic needle inserted into it.

FIG. 5 is a cross-sectional view of the assay unit of FIG. 1 with astandard laboratory pipette holder inserted therein.

FIG. 6 is a cross-sectional view of the assay unit of FIG. 1 with one ofthe sealing surface areas being used as a sample cup.

FIG. 7 is a cross-sectional view of the assay unit of FIG. 1 insertedinto a aspiration pump in action.

FIG. 8 is a schematic view showing the assay unit of FIG. 1 connected toa substrate pump for its input with its output empting into a microplatewell.

FIG. 9 is a view similar to FIG. 8 with an optical detection on theoutput of the assay unit based on absorbance.

FIG. 10 is a view similar to FIG. 8 with an optical detection on theoutput of the assay unit based on fluorescence.

FIG. 11 is a graphical representation of some data obtained using theassay unit of the present invention, this data showing typical enzymeabsorbance signal resulting from substrate being pumped through an assayunit containing bound enzyme conjugate at varying flow rates.

FIG. 12 is a graphical representation of data showing absorbance (OD)signals at two substrate flow rates as a function of sampleconcentration for direct ELISA assays run using the assay unit.

FIG. 13 is a graphical representation similar to FIG. 12 with reducedconcentrations.

FIG. 14 is a graphical representation showing the absorbance (OD)signals at 30 nL/sec substrate flow rate as a function of sample massfor sandwich ELISA assays run in the separation unit.

FIG. 15 is a cross-sectional view showing the assay unit with a needleinserted into its input shown pumping buffer through the unit todisplace any air.

FIG. 16 is a cross-sectional view showing the assay unit partiallyfilled with liquid being inserted into a partially liquid-filled inputto the aspiration pump.

FIG. 17 is a graphical representation of data showing absorbance (OD)signals of repeats of the same sample for a direct ELISA assay with andwithout air entrapped in the assay unit.

FIG. 18 is a cross-sectional view through an aspiration or piston pumpconstructed in accordance with another aspect of the present invention.

FIG. 19 is an enlarged cross-sectional view showing an assay unitinserted into the pump of FIG. 18.

FIG. 20 is a cross-sectional view of a multi-channel pump system for usewith an array of the assay units.

FIGS. 21 through 28 are cross-sectional views illustrating steps in theuse of the pump and assay unit of the present invention.

FIG. 29 is a cross-sectional illustration of two nested assay units.

FIG. 30 shows a cross-sectional view of an assay unit with a needleinserted pumping eluent through the bed of the assay unit.

FIG. 31 shows the fraction of an antibody-enzyme conjugate captured inassay units as a function of the residence time in the unit fordifferent particle diameters of the packing material.

FIG. 32 shows calculations from an engineering design model based on thedata in FIG. 31 for the maximum flow rate for 95% capture through anassay unit and the resulting assay time, as a function of the packingparticle diameter.

DETAILED DESCRIPTION OF THE INVENTION

This invention has four related aspects. One aspect relates to thephysical design of the assay unit. A second aspect is the method ofoperation for the assay unit in an immunoassay, particularly taking intoaccount the removal of entrapped air in order to provide reproducibleresults. A third aspect is the design of an aspiration pump for optimaluse with the assay unit to perform the method. A final aspect is theselection of the assay unit packed bed geometry (diameter and length)and particle size for optimal operation of the design in the method.These aspects will be first introduced generally and then described indetail.

Separation unit design.

The assay unit includes a packed bed of adsorbent particles containedwithin a cylindrical or frustum-shaped chamber by inlet and outletscreens or filters. The inlet to the packed bed includes a series ofthree open frustum-shaped open chambers, forming a tapered “cup”. Thesuccessive chambers permits the unit to accept input from devices in avariety of sizes.

The chamber immediately adjacent to the inlet screen of the packed bedforms an “inlet seal” area, designed to reversibly connect by simpleinsertion to standard 10 and 200 μL pipette tips, as well as severalstandard gauge hypodermic needles or similar tubes, reliably forming atight, relatively high pressure seal. The next chamber out is a “samplecup” designed to contain small volumes of samples or reagent to beintroduced into the packed bed. The outer chamber is designed to mateand seal with the distal end of a standard 200 μL pipette holder orsimilarly-dimensioned device.

The outlet of the assay unit is also frustum-shaped, with an outerprofile similar to the distal end of a standard 200 μL pipette tip. Theoutlet screen of the packed bed is located very close to the outletport. This shape enables liquids flowing out of the assay unit to bedeposited as small droplets on precise spots, such as MALDI-MS targetplates. This shape also enables the outlet of the assay unit to beeasily connected by simple insertion into a port with the same profileas the “inlet seal” of the assay unit itself. The overall geometry ofthe assay unit also enables the outlet of one unit to connect to theinlet seal of a second unit, enabling liquids to be transferredefficiently from one unit to another.

Method of Operating the Assay Unit

In general operation, the outlet of the assay unit is inserted into aninlet connected to a pumping system (preferably an aspiration pump)capable of aspirating liquids through the outlet at a controlled flowrate. Reagents, samples or washing solutions are measured and dispensedinto the sample cup chamber of the assay unit using standard manualpipettes or automated liquid handling systems. The measured volume ofliquid is then pulled through the bed using the aspiration pump. Inaddition, it is possible to insert the distal end of the pipette tipused to measure the liquid directly into the inlet seal of the assayunit and leave the pipette tip behind as a “reservoir”. This mode ofoperation is useful either for very small volumes to prevent transferlosses, or for larger volumes to extend the volume of the sample cup.

After all of the liquids required for each of the assay steps are pumpedthrough the packed bed in this manner, an analytical measurement orresult may be obtained in either one of two different general methods.In the first method, bound enzyme is measured by pumping a solution ofenzyme substrate through a controlled flow rate, and substrate isconverted to enzyme product in the packed bed at a rate which is afunction of both the substrate flow rate and the amount of enzyme boundto the bed. The resulting enzyme product (and therefore bound enzymereagent) concentration may be determined by collecting the productsolution from the outlet of the assay unit in a microplate well andusing a standard optical plate reader. Alternatively, the productconcentration may be measured by connecting the assay unit outlet to anoptical detector comprising a flow cell optically coupled to anappropriate light source and detector to measure the optical absorbance,fluorescence or chemiluminescence of the liquid emerging from the assayunit. In the second method of measurement, bound molecules, which may belabeled with a fluorescent marker, are eluted from the packed bed bypumping a solution through the bed that detaches the bound moleculesfrom the binding. As is well know in the art, this is often done by saltor acid based elution solutions. The output from the assay unit is thenmeasured optically, by fluorescent sensing, to determine the amount ofthe labeled molecule that was bound in the packed bed.

An advantageous part of the method of operation is a technique to removeair that remains entrapped in the packed bed after each aliquot ofliquid has been aspirated through the assay unit. If the entrapped airbubbles are not removed, the surface area of the bed is reduced by avariable amount and the liquid flow path through the bed is disrupted,causing random variation in the final assay results. The entrapped airmay removed by flushing the packed bed at a high flow rate with water ora wash buffer solution, either by positive displacement pumping oraspiration through the inlet seal or the outlet seal. The outlet of theliquid-filled bed assay unit can be inserted into a connection portwhich is partially filled with liquid to make a connection withoutentrapping further air. The inlet seal of the assay unit is left filledwith liquid so that when a new sample or reagent liquid aliquot isdispensed into the sample cup, the liquids “merge” without entrapment ofair bubbles. When this technique is employed between assay steps, thereproducibility of the assay system is dramatically improved.

When used for micro-volume sample preparation, the method of operationis similar. For the final step, instead of pumping enzyme substratethrough the assay unit, an eluent solution (such as acid or salt) ispumped through the packed bed and the eluate liquid is collected fromthe assay unit outlet onto a surface or into a collection tube. It isalso possible to operate the system with two assay units connectedtogether so that liquid emerging from the outlet of one unit istransferred directly into the inlet of the second unit. This mode ofoperation is useful for multi-step separations or coupling of reactionswith enzymes immobilized on the packed bed to downstream separationsteps.

Aspiration Pump Design

The assay unit is designed so that reagents and samples can be added tothe sample cup using standard manual pipettes or robotic liquid handlingdevices, then are pulled or aspirated through the packed bed by a pumpconnected to the assay unit outlet. A number of different approaches forthis pumping could be used, but a syringe or piston type pump provides agood combination of relative simplicity, excellent flow and volumeprecision and low cost. One problem encountered, however, is that anybubbles present in any tubing connecting the pump to the assay unitcause the loss of precise control of the flow rate. Valves are alsoproblematic because of the very low flow rates involved and thepossibility of bubble entrapment or slow leakage, especially at negativepressure.

To overcome this limitation, a piston-type pump for use in aspirationhas been designed so that the outlet of the assay unit inserts directlyinto a frustum-shaped inlet port very closely connected to the inlet ofthe piston cylinder. The piston is designed so that there is very littledead volume between the assay unit and the piston when the piston isfully inserted, in order to minimize the possibility of entrapped airbubbles. Once the aliquot of liquid has been aspirated completelythrough the packed bed, the assay unit is lifted out of the aspirationpump inlet port and the cylinder is emptied by moving the piston upward.A suction port entering the side of the pump inlet port located abovethe sealing point between the inlet port and the assay unit outlet pullsthe expelled liquid out into a waste reservoir held under vacuum. Asecond port entering the side of the pump inlet may also be used is usedto introduce liquids for washing the pump inlet, cylinder, piston andassay unit tip between cycles.

Bed Geometry

Working with this apparatus and this method has revealed a “window” ofthe combination of adsorbent particle type and size and packed bedgeometry (diameter and length) which results in optimal operation inimmunoassay applications. The combination comprises non-porous particleswith an average diameter in the range of 20 to 150 μm. The particlesmust have an appropriate surface chemistry for irreversible binding ofactive antibodies, antigens or other coating reagents. The bed diameteris in the range the outer diameter of the distal end of standard pipettetips (0.5 to 1.0 mm) in order to meet the design constraints for theinlet seal and outlet of the assay unit itself. The bed length isselected to give a total adsorbent particle surface area in the packedbed of 0.5 to 2 square cm. For particles with a diameter in the range of20 to 150 μm this gives bed lengths from 1 to 50 mm.

These elements will now be described in detail.

In FIG. 1, reference numeral 1 is directed to the assay unit of thepresent invention. FIG. 1 depicts the general layout and key features ofthe assay unit or assay unit 1, comprising a very small volume packedbed of particles 2. The packed bed 2 is contained within a cylindricalor frusto-conical reaction chamber or bed having a defined inlet andoutlet sealed by a pair of porous bed supports 3. These bed supports mayconsist of any of a wide range of woven or non-woven screens, filters ormembranes made from polymer, metal or paper with an average pore sizewhich will contain the adsorbent particles. The outlet 4 from the packedbed chamber is located close to the distal end of the assay unit 1, andthe shape of the outside of the distal end is designed to be identicalto the outside of the distal end of a standard 200 μL pipette tip, witha final diameter of less than 1 mm.

The inlet of the packed bed chamber is shaped so as to have threedistinct frustum-shaped surfaces forming seals to which various inputdevices can mate in fluid-tight fashion. The surfaces are formed inseries, forming a tapered receptacle serving as a sample cup for inputreagents. Closest to the inlet of the packed bed 2 is the smallestsealing surface 5, which is frusto-conical in shape and has an inletdiameter 6, an outlet diameter 7 and a length 8 carefully defined toenable standard small volume pipette tips and standard gauge hypodermicneedles and tubing to connect in a fluid-tight seal by simple insertion.Above the sealing surface 5 is another frusto-conical chamber serving asa sample cup 9, which has a volume designed to hold typical requiredamounts of samples or reagents, typically ranging from 5 to 100 μL. Thefinal sealing surface 10, adjacent to the proximal end of the assay unit1 is another frusto-conical sealing surface sized to fit and seal on thedistal end of a standard 200 μL laboratory pipette (i.e. is shapedidentically to the proximal end of a standard 200 μL pipette tip).

The dimensions of the inlet seal 5 are critical for enabling the inletof the packed bed 2 to be in fluid-tight connection to a variety ofdifferent standard fluid handling devices. The inlet diameter 6 isselected so that the distal end of a standard 200 μL pipette tip 11 willjust fit into the upper portion of the inlet seal 5, as shown in FIG. 2.This inlet diameter 6 is at least 1 mm and preferably in the range of1.2 to 1.5 mm. The outlet diameter 7 is selected so that the distal endof a standard 10 μL pipette tip 12 will fit and just be prevented fromtouching the inlet retention means 3, as shown in FIG. 3. This outletdiameter 7 is less than 0.8 mm and preferably in the range of 0.7 to0.75 mm. The inlet seal length 8 determines the angle between the wallsof the inlet seal 5. This angle must be slightly greater than the outerangle of the standard pipette tips 11 or 12 in order to form a reliableseal on the very end of the tips to prevent dead spaces and holdup ofliquids between the tip 11 or 12 and the inlet sealing surface 5. Forcommercially available tips, the angle between the center axis and thewall of the inlet seal 5 should be in the range of 5 to 7 degrees. Withthese dimensions, the inlet seal 5 will also seal as shown in FIG. 4 tostandard straight wall tubing 13 between 0.75 and 1 mm OD, including 19to 21 gauge hypodermic needles.

This type of sealing mechanism is highly reliable, with gentle forcealong the axis of the assay unit 1 being all that is required to make orbreak the seal. Seals can be easily be made by automated roboticsystems, which aids in automating the entire assay process. Because ofthe very small diameters involved, the seals are capable of pressures inexcess of 5 bar, even with just the friction of the interfering taperfit.

The uppermost or hub 10 chamber of the assay unit enables the device tobe placed on the end of a standard 200 μL laboratory pipette holder 14or any other device with an identical profile, as shown in FIG. 5. Inthis mode, the assay unit 1 could be operated like other pipettetip-like solid phase extraction devices using air displacement to pullliquids in or push them out. However, it has been found that this meansof operation is not as useful for the highly controlled application ofsamples and reagents required for precise assays. In the system of thepresent invention, the hub 10 feature is primarily used to enable adisposable pipette tip head on a robotic liquid handling system to pickup an assay unit 1 and place it in the proper location. Once inposition, the normal tip ejection mechanism is used to release the unit1 from the robotic head.

One important objective of the present invention is to functionallyseparate the steps of measuring and dispensing aliquots of samples,reagents, wash solutions and other liquids into the assay unit 1 fromthe step of pumping the liquid aliquots through the packed bed 2. Thisfunctional separation confers a number of significant advantages. Mostconventional manual or automated liquid handling systems are capable ofmeasuring and depositing a precise and accurate volume into a givenlocation, but are not generally capable of providing tightly controlledflow rates because they operate using air displacement or airsegmentation. However, any of these systems can be used for themeasurement/loading step, imparting a great deal of flexibility to thedesign and operation of the required instrumentation, as well as makingautomation easier using standard components. In addition, a simple andinexpensive single channel liquid handling system can be used formeasurement and dispensing in combination with a multi-channel flowsystem to obtain high assay throughput.

This mode of operation is illustrated in FIGS. 6 and 7. The outlet ofthe assay unit 1 is inserted into an aspiration pump 15. This aspirationpump can be any pumping system capable of pulling liquids out of theassay unit 1 at a controlled and reproducible flow rate, independent ofthe flow resistance. Types of aspiration pumps could include positivedisplacement pumps such as piston or syringe pumps, peristaltic pumps orgear pumps. The aspiration pump 15 might also be a vacuum sourceconnected to the assay unit 1 through a rapid acting solenoid valve orcontrol valve to regulate the flow. The inlet port of the aspirationpump 15 is shaped similarly with dimensions similar to the inlet seal 5of the assay unit 1 itself. The aspiration pump 15 can have a singlepumping channel or have multiple pumping channels for operating severalassay units 1 in parallel, either at the same flow rate or at differentflow rates. A multi-channel aspiration pump 15 is advantageous forincreasing the assay throughput of the system.

As shown in FIG. 6, an aliquot of sample or reagent liquid 16 isdispensed into sample cup 9 of the assay unit 1 with the aspiration pump15 turned off. As shown in FIG. 7, the aspiration pump 15 is then turnedon and the liquid aliquot 16 is pulled through the assay unit 1 at acontrolled flow rate. Generally, in order to insure that the completemeasured volume goes through the assay unit 1, the liquid is pulledthrough completely, which then pulls air into the packed bed 2. For animmunoassay, a series of reagents, samples and wash solutions are putthrough the assay unit 1 in the proper sequence using this mechanism.

The final reagent is typically an antibody or antigen conjugated to anenzyme, which serves as the label for measurement. Detection andmeasurement of the level of enzyme conjugate bound to the assay unit 1at the end of the assay steps is done by transferring the unit to adetection station, as shown in FIGS. 8 to 10. The inlet seal 5 of theassay unit 1 is connected via tubing 13 to a pump 17 which is filledwith a solution of substrate for the enzyme. The substrate pump 17 maybe any type of high precision positive displacement pump, including apiston or syringe pump, peristaltic pump or gear pump. Because theoptical signal depends critically upon the substrate flow rate,precision, stability and lack of pulsation are critical characteristicsof the substrate pump 17. In general, piston-type pumps give the bestperformance.

One mode of detection, shown in FIG. 8, is to collect the reactedproduct from the outlet of the assay unit 1 into the well 18 of aconventional microplate. Multiple assay units can be run and collectedthis way in parallel using a multi-channel substrate pump. Once a knownvolume of product solution is collected in the well, the microplate maybe read in a conventional optical plate reader instrument, in order todetermine the optical absorbance, fluorescence or chemiluminescence.This method has the advantage of utilizing widely available plate readerinstrumentation.

A second mode of detection is to insert the outlet of the assay unit 1into the inlet port of an optical flow cell 19 set up to read either theabsorbance (FIG. 9) or fluorescence (FIG. 10) of the liquid entering thecell. A transparent illumination window 20, optionally with an opticalfiber, is used to connect the flow cell to an appropriate light source21, which may be monochromatic or polychromatic. For optical absorbancemeasurements, a second measurement window 22 is placed at the other endof the illuminated flow path facing the illumination window 20. Themeasurement window 22 is connected optically (optionally by a fiber) toan optical detector 23, which may be a simple photodetector if amonochromatic light source is used, or a spectrometer if a monochromaticlight source is used. The detector 23 is used to measure the absorbanceof the liquid in the flow cell at a particular wavelength. In analternative configuration (FIG. 10), the measurement window 24 is placedat right angles to the beam from the illumination window 20 and isconnected optically to an optical detector 25. This configuration isused for fluorescence measurements. If the light source is turned off,either configuration may be used for chemiluminescence measurements. Theuse of a flow cell has the advantages that a much smaller volume may beread, decreasing the readout time, and dynamic changes in the output asa function of operating conditions may be observed.

FIG. 11 shows a typical absorbance output from a system similar to thatshown in FIG. 9. In this example, the packed bed contained 20 μmdiameter non porous particles of polystyrene-divinylbenzene. The beddimensions were 0.8 mm diameter and 5.5 mm long (3 μL bed volume). Theassay unit was pumped with the following reagents in sequence: VolumeReagent Flow Rate 10 μL Coating antigen - 500 μg/mL bovine IgG 6 μL/min(Sigma) in 50 mM carbonate pH 9.6 10 μL Blocker - 10 mg/mL fish gelatinprotein 24 μL/min (Sigma) in 50 mM Tris, 0.14 M NaCl, 0.05% Tween 20 pH8.0 10 μL Conjugate sample -sheep anti-bovine IgG 3 μL/min conjugated tohorseradish peroxidase (Bethyl Labs) diluted to various concen- trationsin Blocker 20 μL Wash - 50 mM Tris, 0.14 M NaCl, 0.05% 500 μL/min Tween20 pH 8.0

This is an example of a “direct ELISA” assay, in which the solid phaseis coated with antigen, non-specific adsorption sites are blocked and asample containing a particular concentration of antibody-enzymeconjugate is applied.

Following the binding reactions, the assay unit was transferred to anabsorbance detector system similar to that shown in FIG. 9 and pumpedwith a substrate solution (0.4 mM tetramethyl benzidine (TMB, Sigma)with 0.15% v/v hydrogen peroxide in 50 mM phosphate-citrate pH 5.0). Theabsorbance was measured at 650 mn. Note that normally the enzymereaction in an ELISA must be “stopped” after a fixed time, usually withacid, in order to obtain a fixed reading. Since the assay unit 1 of thecurrent invention is a flow-through system, the substrate stops reactingas soon as it leaves the packed bed 2, and no separate stopping step isnecessary.

FIG. 11 shows the typical output signal. When substrate pumping begins,a short transient is experienced due to the refractive index differencebetween the final wash solution and the substrate solution. The signalthen increases until it reaches a steady state at a particular flowrate. The time required to reach steady state will depend upon theliquid volumes in the assay unit 1 and the flow cell 18 as well as thesubstrate flow rate. The difference in optical density (OD) between thebaseline and the steady state is the readout used to determine thesample concentration.

It may be observed in FIG. 11 that the signal increases as the substrateflow rate decreases. This feature enables the analytical sensitivity ofthe system to be adjusted by changing the substrate flow rate. Indeed,multiple flow rates may be run for each assay on the same sample,enabling multiple standard curves to be developed and a broader assayrange to be covered. FIGS. 12 and 13 show plots at two different scalesof the steady state OD signals at two different substrate flow ratesover a range of different conjugate concentrations in the sample usingthe same assay protocol presented for FIG. 11. Each sample was measuredat the two flow rates, producing two different curves. At 30 nL/secsubstrate flow the assay is considerably more sensitive to lowerconcentrations (FIG. 13), but because bot the reaction and the detectorcan saturate, the assay becomes nonlinear above around 200 ng/mLconjugate (FIG. 12). However, the signal at 100 nL/sec substrate flow islinear up to 1000 ng/mL, providing an extended linear range for theassay.

FIG. 14 shows results using the system for a “sandwich” format ELISAusing the system of the present invention. In this example, the packedbed contained 20 μm diameter non porous particles ofpolystyrene-divinylbenzene. The bed dimensions were 0.8 mm diameter and5.5 mm long (3 μL bed volume). The assay unit was pumped with thefollowing reagents in sequence: Volume Reagent Flow Rate 25 μL Coatingantibody - 100 μg/mL sheep anti- 6 μL/min bovine IgG (Bethyl Labs) in 50mM carbonate pH 9.7 10 μL Blocker - 10 mg/mL fish gelatin protein 24μL/min (Sigma) in 50 mM Tris, 0.14 M NaCl, 0.05% Tween 20 pH 8.0 10 μLAntigen samples - Dilution of reference 3 μL/min bovine serum with 28mg/mL bovine IgG in Blocker 10 μL Conjugate - 5 μg/mL sheep anti-bovineIgG 3 μL/min conjugated to horseradish peroxidase (Bethyl Labs) inBlocker 30 μL Wash - 50 mM Tris, 0.14 M NaCl, 0.05% 500 μL/min Tween 20pH 8.0

The assay units were run in the same system used for FIGS. 11-13, withthe same enzyme substrate solution. The total time for all of the assaysteps required was 11.2 minutes. The data showed good linearity over therange tested (R2=0.993).

Note that FIG. 14 is plotted as OD signal vs. mass of antigen ratherthan the conventional concentration to illustrate an important featureof the assay method of the present invention. Because the solid phase isa flow-through packed bed as opposed to a standard microplate well, avery wide range of different sample volumes may be used. The volume in amicroplate well is limited to a very small range (typically 50 to 100 μLfor a 96-well plate) in order to expose the sample to the entire coatedbinding surface. In the assay unit of the present invention, the bindingsurface is compressed into a small packed bed with a very small liquidvolume (typically 0.5 to 2 μL). Target molecules in the sample bind tothe bed as they flow through, and thus the bed serves as a“concentrator” for the sample. The two sample results highlighted inFIG. 14 had the same mass of target antigen (0.3125 ng), differing involume and concentration (10 μL of 31.25 ng/mL vs. 40 μL of 7.8 ng/mL),yet giving the same signal.

Reproducibility or precision is a critical element of any analyticalmethod. Conventional microplate-based ELISA methods suffer from poorreproducibility (C.V.'s of 10 to 30% are typical) for a number ofreasons. One is that it is difficult to precisely control the timing ofthe reagent or sample addition into all of the wells on the plate sothat the incubation times for all of individual samples in the set areprecisely the same for each well. Pipetting technique can also becritical and difficult to control reproducibly. During the incubationsteps mixing from jarring, moving or vibrating the plates can causevariable results, as can temperature changes in the incubationenvironment. Even varying conditions between the outer and inner wellsof a plate can give rise to variability in the final results.

The assay system of the present invention can substantially reduce oreliminate these sources of variability. Reagent addition (done at acontrolled flow rate instead of by incubation) can be very reproduciblewith proper design of the pumps used. The issue of variable mixing isalso dealt with by the use of flow through a packed bed. The mostcritical parameters controlling reproducibility are the measurement ofthe sample volume (all other reagents are added in excess, so volumecontrol for them is less critical) and the flow rate of the substrateaddition. These can be easily controlled to a precision of well under 5%using standard instrumentation.

One unexpected potential source of assay variability in the presentinvention proved to be the entrapment of air bubbles in the bed. Oneconsequence of the use of an aspiration pump 15 in order to functionallyseparate the volume measurement and dispensing from the flow loading ofan aliquot through the assay unit 1 (as illustrated in FIGS. 6 and 7) isthat in order to deliver the entire aliquot of liquid 16 into the packedbed 2, it is necessary to pull air into the bed after each aliquot. Ithas been discovered that if the next aliquot of liquid is simply added,in the manner shown in FIG. 6, air bubbles of random volume and positionmay remain behind in the bed. These entrapped bubbles effectively blockpart of the bed from exposure to the liquid, and also disrupt the flowpattern within the bed, which can cause significant variability in thefinal assay results.

A solution to this problem is illustrated in FIGS. 15 and 16. A tube 13connects the inlet seal 5 of the assay unit 1 to a syringe or pumpfilled with a wash solution. The wash solution is pumped through thepacked bed 2 at a flow rate and volume high enough to dislodge andremove entrapped air through the outlet 4. It is also possible to removeair bubbles by filling the sample cup 9 with wash buffer and rapidlyaspirating the liquid through the packed bed via a pump or syringeconnected to the outlet 4.

The flow rate required for complete removal of entrapped air bubblesfrom the packed bed depends upon the diameter and length of the bed andthe particle diameter. Generally, higher flow rates are required forlarger beds and larger particle diameters. Testing with a 0.75 mmdiameter, 3 μL bed of non-porous polystyrene-divinylbenzene beadsindicated that volumes of 20 to 50 μL were adequate for all beaddiameters. The flow rate required for complete clearance of bubbles wasat least 15 μL/sec for 20 μm diameter particles, 25 μL/sec for 50 μmdiameter particles and greater than 50 μL/sec for 120 μm diameterparticles. A simple way to provide these high flow rates is to connectthe assay unit inlet seal 5 or outlet 4 to a spring, air orsolenoid-actuated syringe that can provide substantial pressure to theliquid during the flushing step.

Once air removal is completed, the inlet seal 5 is left full of liquid26 so that additional liquid added to the sample cup 9 will “merge”without entrapping a bubble. The liquid-filled assay unit may also beinserted into inlet ports 27 on the aspiration pump or flow cell withoutentrapping bubbles by having the port partially filled with liquid sothat an initial “liquid seal” is formed before the solid seal iscompleted by insertion of the assay unit outlet 4.

FIG. 17 illustrates the effect of this air removal procedure on theassay reproducibility. In these experiments the packed bed contained 20μm diameter non porous particles of polystyrene-divinylbenzene. The beddimensions were 0.8 mm diameter and 5.5 mm long (3 μL bed volume). Theassay unit was pumped with the following reagents in sequence: VolumeReagent Flow Rate 10 μL Coating antigen - 500 μg/mL bovine IgG 6 μL/min(Sigma) in 50 mM carbonate pH 9.6 10 μL Blocker - 10 mg/mL fish gelatinprotein 24 μL/min  (Sigma) in 50 mM Tris, 0.14 M NaCl, 0.05% Tween 20 pH8.0 10 μL Conjugate sample - 250 ng/mL sheep anti- 3 μL/min bovine IgGconjugated to horseradish peroxidase (Bethyl Labs) in Blocker 20 μLWash - 50 mM Tris, 0.14 M NaCl, 0.05% Tween varied 20 pH 8.O

The assay units were run in the system illustrated in FIGS. 11 to 13,with the same enzyme substrate solution. In the samples run without airremoval, the assay steps were run as shown in sequence, and the finalwash step was run at 24 μL/min. In the samples run with air removal, theassay unit was flushed after each step with 20 μL of wash buffer at 8μL/sec (480 μL/min), and the final wash step was run at this same highflow rate. Each procedure was repeated with 5 identical samples. FIG. 17shows the results from these examples. Without air removal, thecoefficient of variation (C.V.) for the 5 runs was 22%, while with airremoval the C.V. was 2%, thus demonstrating the dramatic improvement inreproducibility achieved by avoiding air entrapment.

An additional potential problem is with the aspiration pump 15 itself.Air bubbles present downstream of the packed bed 2 can cause variationsin the flow rate. Since the major flow resistance is from the packed bed2, the pressure downstream must be lower than atmospheric to cause flow,and bubbles trapped downstream will therefore expand in order toequalize the pressure. This can be a particular problem when theair-liquid interface passes through porous “barriers” created by the bedsupport 3 and the packed bed 2 as surface tension forces cause a anincreased pressure drop required for the interface to move through thebarrier. Once the air passes through the barrier, the required pressuredrop for flow decreases again. If bubbles are present downstream, theymust then expand to enable flow to pass through the barrier (causingflow to temporarily slow or stop), then will contract once the barrieris passed (causing flow to suddenly increase until the bubblere-equilibrates). For this reason, it is critical to keep bubbles out ofthe liquid volumes downstream of the packed bed 2 as well.

Although a number of different types of pumps may be used for aspirationas described above, it has been found that a piston-type pump of thedesign shown in FIGS. 18 and 19 has a number of very importantadvantages. In this pump, a piston cylinder body 28 is inserted, or isformed integrally, into a pump block 29 which houses a reciprocatingpiston 30. The pump block also has formed in it a frustro-conical shapedinlet port 31, larger at its upper end, which designed to seal with theoutlet tip 4 of an assay unit 1, and open at its smaller lower endconnecting into the interior of the cylinder body 28. The inlet port 31opens into an inverted frustum shaped passage designed to fit tightlywith the top of the piston 30. In addition, the inlet port 31 has asmall lower side port 32 which connects the upper frustum to a smallplenum 33. Optionally, the pump may also include a second upper sideport 34 located above the lower side port 32 connecting the upperfrustum to a second small plenum 35. As shown in FIG. 19; when the assayunit 1 is fully inserted into the inlet port 31 and the piston 30 isfully inserted, the sealing point 36 is located between the side port 32and the top of the piston 30, thus closing off the fluid connectionbetween them and permitting only a minimal liquid volume for possiblebubble entrapment. In effect, the sealing point of the assay unit itselfserves as a valve, directing flow caused by the piston movement eitherthrough the packed bed or to the side ports of the pump.

As shown in FIG. 20, it is possible to create a multi-channel array of anumber of these pistons 30, inlet ports 31 and side ports 32 and 34 incombination, with the pistons 30 connected to a common drive mechanismplate 37, in turn connected to a drive mechanism 38 which moves all ofthe pistons up or down at the same time. In order to make the systemcompatible with common liquid handling devices, it is advantageous toarrange the cylinders and inlet ports in linear arrays with the ports on9 mm centers. In addition to the single linear array shown in thefigure, it is also possible to have a two-dimensional array, for examplewith 96 channels arranged in 8 rows of 12, similar in layout to astandard 96-well microplate. The first side ports 32 are connected to acommon plenum 33, which is in turn connected through a valve 40 to aclosed collection reservoir 41 connected to a vacuum pump or source 42.When the valve 40 is opened, any liquid in the inlet ports 31 above thelower side ports 32 will be removed by suction through the plenum 33into the collection reservoir 41.

Similarly the second side ports 34 are connected to a second plenum 35which is in turn connected to a pump 43 and one or more reservoirs 44 ofliquid used for washing out the inlet ports 31. When pump 43 isactivated, wash liquid from reservoir 44 is pumped into all of the inletports 31 simultaneously. The system is also equipped with a locatorplate 39 through which the assay units may be inserted and held withoutmoving relative to the plate. The plate may be driven up or down by anactuator, causing all of the assay units 1 to be moved up or down atonce relative to the aspiration pump block 29.

The operating cycle of this pump is shown in FIGS. 21 to 28. In FIG. 21,an assay unit 1 is inserted into the pump and a pipette or other liquidhandling device is used to deposit a precisely measured volume of liquid45 (reagent, buffer, sample, etc.) into the sample cup of the assay unit1. To start the flow (FIG. 22), the piston 30 is moved downward at acontrolled speed by the pump drive, which in turn pulls the liquid 45through the assay unit 1 packed bed at a controlled flow rate. In orderto insure that all of the liquid 45 is exposed to all of the packed bedfor a controlled time, the piston 30 is pulled down far enough (FIG. 23)so that air is pulled through the assay unit 1 packed bed. The assayunit 1 is then moved upward (FIG. 24) by the locator plate 39 so thatthe seal with the inlet port is broken, making a fluid connectionbetween the top of piston 30, the side port 32 and the plenum 33. Thepiston is then moved upward to the top of its stroke to expel the liquid45 upward. Suction applied to the plenum 33 pulls the liquid out theside port 32 where it is carried away through the plenum 33 to a wastecollection reservoir.

As shown in FIG. 25, the assay unit 1 may be moved downward until itsoutlet tip 4 is located between the first side port 34 and the secondside port 32. In this position, wash liquid 48 may be pumped in throughthe upper plenum 35, through the upper side port 34 and sucked outthrough the lower side port 32 into the lower plenum 33. This flow ofliquid can be used to wash out the inlet port 31 as well as the outsideof the assay unit 1. As shown in FIG. 26, the assay unit can bewithdrawn and the washing flow continued while the piston 30 is moved upand down in order to wash the piston and cylinder.

The small amount of wash liquid 48 remaining in the inlet port below theside port 32 is used to form a “liquid seal” (as illustrated in FIG. 16)when the assay unit 1 is reinserted to complete the cycle (FIG. 27). Ifa small amount of wash liquid is held in the cylinder by keeping thepiston 30 partially withdrawn during this step, once the seal with theassay unit 1 is made, the piston 30 can be driven up quickly, expellingthe air from the packed bed and leaving a small amount of wash liquid 48in the inlet seal to start the next cycle.

The same assay unit of the present invention can also be used in amicro-preparative mode to purify particular molecules of interest forother micro-scale analytical techniques such as mass spectroscopy. Inthis mode, packed bed contains any of a number of different particulateadsorbents (including but not limited to porous or non-porous particles,made of materials such as polystyrene-divinylbenzene, polyacrylamide,agarose, cellulose, silica, alumina, zirconia, composites thereof andthe like) with immobilized binding molecules (including but not limitedto antibodies, antigens, nucleic acids, hormones, cytokines, receptors,enzymes, and the like) or other selective surface chemistries (includingbut not limited to ion exchange, reversed phase, hydrophobicinteraction, gel filtration, affinity chromatography, mimetic ligandchromatography, metal chelate chromatography and the like). Samplescontaining the target molecules are passed through the packed bed andbind to the selective adsorbent particles and non-binding contaminantsare washed away. Then the bound target is eluted using, for example,acid or a salt solution, for collection into a tube or on a surface spotfor further analytical processing. It is also possible to immobilize anenzyme (including but not limited to proteases, kinases or glycosidases)to the packed bed to enable very rapid selective digestion or otherprocessing of the target molecule as it passes through the packed bed ata controlled flow rate prior to further analysis.

Two additional modes of operation are useful for these micro-preparativeseparations. FIG. 29 shows two assay units (1 and 49) connected togetherby the outlet of the first unit 4 inserted into the inlet seal 50 of thesecond unit 49. This mode of operation is useful for severalapplications. One would be to combine multiple separation steps on anautomated system. For example, the first assay unit 1 could contain anion exchange packing to selectively bind and purify the target from acomplex sample such as blood serum or cell culture supernatant. Elutionfrom this packing is through the use of a high concentration of salt,which is not compatible with mass spectrometry. If the second assay unit49 contains a reversed-phase packing, when the target is eluted from thefirst unit 1 into the second unit 49 it will be bound on the reversedphase packing. After the units are decoupled the salt can be washedaway, and the target eluted from the second unit 28 using an organicsolvent solution that is compatible with the mass spectrometer.

A second type of application for the mode of operation illustrated inFIG. 29 is the use of an immobilized protease, such as trypsin, in thefirst unit 1. During passage of a sample aliquot through the first unit1, the proteins present would be digested by the immobilized enzyme intodefined peptides. By using immobilized enzyme, a much higher amount ofenzyme can be used than is normally employed in the solution phase,giving rise to a faster digestion with no chance of autolysis productsfrom the enzyme contaminating the analysis. If a reversed phase packingis used in the second unit 49, the digested peptides would be capturedand concentrated, and any salt required in the digestion buffer would beremoved by washing after the units are decoupled. The peptides couldthen be eluted in an organic solvent solution that is compatible withthe mass spectrometer.

FIG. 30 illustrates the preferred mode of elution from the assay unit 1for micro-preparative applications. Eluent solution is pumped through atube 13 connected to the inlet seal 5 of the assay unit 1. After passingthrough the packed bed 2, the eluate 51 emerges from the outlet tip 4 asa small droplet. The eluate can be collected in very small volumes (lessthan 1 μL is possible) by gently touching the outlet tip 4 with thehanging droplet onto a surface 52, which may be a MALDI MS target plate,test tube, microplate well, electrophoresis gel well, etc.Alternatively, the outlet tip 4 could be inserted directly into theinlet port of an electrospray ionization mass spectrometer and theeluted product analyzed directly by pumping into the instrument at acontrolled flow rate.

The design of the packed bed of the assay unit, including the diameterand length of the bed and the type and diameter of the adsorbentparticles, is critical for optimal operation in the immunoassayapplication. With a standard microplate-type ELISA, each of the reagentsand samples are incubated in the well for a set period of time in orderto expose the molecules in the solution to the binding molecules coatedon the surface of the wall of the well. Although all of the molecules inthe well are available for binding, the only way they can reach the wallis through molecular diffusion, which is rather slow for large moleculessuch as proteins. Thus each step requires an incubation period rangingtypically from half an hour to overnight or longer to reach the bindinglevel desired. Often the time required to complete binding equilibriumis impractically long and therefore shorter times are used, preventingthe binding reaction from going to completion. This causes the assayresults to vary significantly depending upon the exact timing,temperature, and mixing events, such as jarring or moving the plate.

By contrast, in the assay unit of the present invention, the bindingsurface is provided by the packed bed of adsorbent particles. Moleculesare exposed to the binding surface by flowing through the packed bed.Because the diffusion path for the molecules had been greatly reduced(to just the spaces between the particles and potentially pores withinthe particles), the time required to reach binding equilibrium isgreatly reduced. This not only makes the assay much faster, but alsosignificantly reduces or eliminates the common sources of assayvariability.

However, although the mass transport in the packed bed is greatlyimproved, the rate of binding of molecules from the solution to thesurface (or to capture) is not infinite. If the flow rate through thebed is too high, molecules will not have a chance to bind and will flowthrough the bed, resulting in incomplete capture. On the other hand, ifthe maximum flow rate for effective capture is too slow, the steps ofthe assay will take too long, reducing the some of the advantages of thesystem described here over conventional methods.

At least two factors determine the maximum flow rate for effectivelycomplete capture—the mass transport from the liquid phase to the solidsurface and the kinetics of the binding reaction itself. Mass transportto the binding surface, in turn, has two major components; transportfrom the bulk liquid to the surface of the adsorbent particles by acombination of convection and diffusion, and transport within the poresof the particles (if any) by diffusion. Many investigators have studiedmass transport in this type of system in the context of chromatography(Kopaciewicz et al, Journal of Chromatography, 409:111 (1987)), and thiswork has shown that the diffusion within the pores is by far the slowerof the two mass transport elements. This can be mitigated to some extentby using porous particles with some very large pores that allowconvective flow through the particles. (Afeyan et al, Journal ofChromatography, 519:1 (1990)) However, intra-particle diffusion can becompletely eliminated by using non-porous particles. (Kalghatgi andHorvath, Journal of Chromatography, 398:335 (1987)). Chromatographicapplications require a relatively high binding surface area per unitvolume in order to have useful capacity, so the non-porous particlesused have typically been very small (1 to 3 μm). With particles thissmall, pressure drops at normal flow rates are very high, requiringspecial high pressure pumps and other equipment.

For the immunoassay application, however, the required surface area isactually quite low. A standard microplate well filled with the normal 50to 100 μL of sample or reagent has a solid phase surface area ofapproximately 1 to 2 square cm. This corresponds to a total amount ofantibody coated on the surface of around 400 to 800 ng. (Cantarero etal, Analytical Biochemistry, 105:375 (1980)) Any surface area largerthan 1 to 2 square cm would result in excessive use of the expensiveantibody or antigen reagents compared to a microplate assay. Because ofthis low surface area requirement, relatively large diameter non-porousparticles can be used in a packed bed for immunoassays, eliminating theproblems with high pressure drops, which is particularly important whenpumping liquids through the packed bed.

In the apparatus described here, non-porous adsorbent particles can beused for immunoassays as the solid phase in the packed bed. Theparticles may be made of a variety of polymers (including but notlimited to polystyrene-divinylbenzene, polyacrylamide, polyvinylchloride and the like) or inorganic materials such as silica, alumina,zirconia or carbon. Antibodies or antigens may be coated on the surfaceby passive adsorption or by covalent coupling. The particles may also becoated with a hydrophilic polymer to prevent non-specific adsorption,which covalently reactive groups are placed to enable covalent couplingof the coating molecules. Coupling may also be through specificnon-covalent binding, such as the streptavidin-biotin system.

The diameter of the packed bed is limited by the physical designconstraints of the assay unit, as described above. The inlet diameter ofthe bed must be approximately the same as the outlet diameter of astandard 10 μL pipette tip, or less than 0.8 mm. The outlet diameter ofthe bed must be less than the diameter of the assay unit itself at theoutlet bed support (typically 1 to 1.2 mm) minus twice the wallthickness of the assay unit (typically 0.2 mm) or approximately 0.6 to0.8 mm. In order to reduce problems with plugging of the bed andminimize the pressure drop, the bed diameter should be as large aspossible.

In the present invention, once the packed bed diameter is set, thepacked bed length is then determined by the combination of the beddiameter and the adsorbent particle diameter in order to give a totaladsorbent surface area in the required range of 1 to 2 square cm. Thesurface area per unit volume for uniform spheres is given by thefollowing equation:a=6(1−ε)Dp

where a is the area per unit volume, ε is the void fraction of the bed(the part of the volume outside the particles) and Dp is the adsorbentparticle diameter. The following table shows the bed dimensions andvolumes as a function of particle size for beds with a diameter of 0.75mm, a void fraction ε of 0.4 and a packing surface area of 2 cm, whichare typical of the present invention: Particle Diameter Bed Length BedVolume Void Volume Dp μm L mm Vbed μL Vo μL 10 1.2 0.6 0.2 20 2.4 1.10.4 30 3.7 1.7 0.7 40 4.9 2.2 0.9 50 6.1 2.8 1.1 75 9.1 4.2 1.7 100 12.25.6 2.2 150 18.3 8.3 3.3

The adsorbent particle diameter is selected in order to maximize theflow rate that can be used and still get effectively complete capture.As mentioned above, the capture rate may be limited either by masstransport to the surface or the kinetics of the binding reaction. Themass transport to the surface is expected to be influenced by theparticle diameter, since the smaller the particles, the smaller thediffusion path in the spaces between the particles and the larger thesurface area exposed per unit volume of liquid in the bed.

In order to demonstrate this effect, an experiment was performed usingbeds of 0.76 mm diameter and 5.1 mm in length packed with 4 differentdiameter ranges of cross-linked, non-porous polystyrene-divinylbenzenebeads. A 10 μL sample of 25 ng/mL sheep anti-bovine IgG conjugated tohorseradish peroxidase (Bethyl Labs) in 50 mM carbonate buffer pH 9.6was passed through the packed bed at a desired flow rate, then washedimmediately out with 200 μL of distilled water. After this, the amountof bound enzyme in the packed bed was measured with a substrate flowrate of 100 nL/sec as described in FIG. 11 to 13.

In this experiment, the binding reaction is simply the hydrophobicadsorption of the antibody-enzyme conjugate to the surface of the beads.This reaction is expected to be mass transport limited rather thanbinding kinetics limited. FIG. 31 shows the results, plotted as the %capture versus the residence time (equal to the void volume of thecolumn (1.02 μL) divided by the flow rate). As the residence increases(i.e. the flow rate decreases), the amount of capture approaches 100%.However, the residence time required for complete capture increased withthe particle diameter as expected. Thus, looking only at the masstransport, smaller particle size would enable faster flow rates forcomplete capture.

However, the other potential factor determining the maximum flow ratefor effective capture is the kinetic on-rate of the binding reactionitself. There is some variation in the on-rate, depending upon the sizeof the antigen and the location of the binding site. If the rate ofbinding is kinetically limited (rather than mass transport limited) themaximum flow rate for effective capture should depend only upon thepacked bed volume and not on the bed geometry or particle diamter.

Experiments were conducted with different packed bed diameters, volumesand particle sizes to confirm these relationships. The reagents used andthe sequence of operation were similar to those shown in FIGS. 11 to 13,with a conjugate sample concentration of 200 ng/mL and a substrate flowrate of 100 nL/sec. The loading flow rate for the conjugate sample stepwas varied, and the final OD was determined at each flow rate. Severaldifferent size columns packed with different amounts of differentadsorbent particle diameters were used. The following table summarizesthe results: Particle Bead Void 95% Capture Diameter Bed ID WeightVolume Flow Rate Residence μm mm mg μl μL/min Time sec 20 0.50 0.58 0.350.9 22 20 0.50 0.69 0.42 1.0 25 20 0.75 1.15 0.70 1.6 26 20 0.75 1.380.84 2.1 24 50 0.75 1.38 0.84 2.0 25

The data show that at least for this particular antibody-antigen bindingreaction, a residence time (void volume divided by flow rate) ofapproximately 25 seconds is required for 95% capture, over a fairlybroad range of bed dimensions and particle diameters. This is muchgreater than the residence times required for the binding reaction forthese diameter particles shown FIG. 31 (5 seconds or less). Thissuggests that the binding reaction on-rate is limiting in this system.

FIG. 32 shows calculations from a mass transport and kinetic model basedon these data. In the model, packed beds with a diameter of 0.75 mm anda binding surface area in the bed of 2 square cm are assumed, as shownin Table 4. The curve for “Transport Limited Flow” shows the maximumflow rate at which there is 95% capture of molecules by a rapidreaction, limited by mass transport, such as binding to the surfaceduring coating or blocking steps of an assay. The maximum flow ratedecreases with increasing particle diameter, as shown by the data inFIG. 1.

The curve for “Kinetics Limited Flow” shows the maximum flow rate atwhich there is 95% capture of molecules for a reaction with a higherrequired residence time (30 seconds in this case), such as theantigen-antibody binding reactions of the sample loading and secondantibody conjugate loading steps of an assay. Somewhat surprisingly, thekinetics limited flow rate increases linearly with increasing particlesize. This is because larger particle diameters require larger packedbeds in order to keep the constant surface area. A larger bed can beoperated at a higher flow rate for the same residence time than asmaller bed.

The curve for “Assay Time” shows the sum of these two effects, assuming10 μL volumes for each of the four main steps in an assay—coating andblocking (transport limited) and sample and conjugate (kineticslimited). Below around 25 μm particle diameter the assay time increasessharply, reaching a minimum around 100 μm and increasing slowly forlarger particle diameters. Mass transport effects dominate above about100 μm particle diameter.

To summarize these considerations, the apparatus described here isoptimally operated for immunoassay applications with a packed bed ofnon-porous particles, with a particular operating “window” of thecombination of bed diameter, bed length and average particle diameter.The bed diameter is constrained by the design of the assay unit to arange of 0.5 to 1.0 mm, preferably in the range of 0.7 to 0.8 mm. Thebed length is then determined by the required bed volume, which is inturn determined by the adsorbent particle diameter such that the totalparticle surface area in the bed is in the range of 0.5 to 2 square cm,preferably close to 2 square cm. The average particle diameter isdetermined by a combination of mass transport and kineticconsiderations, and is in the range of 20 to 150 μm, most preferably inthe range of 40 to 100 μm.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is understood that certain adaptations of theinvention are a matter of routine optimization for those skilled in theart, and can be implemented without departing from the spirit of theinvention, or the scope of the appended claims.

1. Apparatus for conducting an immunoassay or selective adsorptionseparation, the apparatus comprising an assay unit having a passageformed in its center to form a packed bed; porous bed supports mountedin the assay unit at opposite ends of the packed bed; solid phasesupport beads located in the packed bed and having a reaction reagentmounted on their surface; an outlet from the unit located below thepacked bed; and two frusto-cylindrical sealing surfaces on the uppersurface of the assay unit above and opening into the packed bed, oneupper sealing surface being larger in diameter than the other lowersealing surface so that two different devices can make fluid-tightcontact with the assay unit without liquid cross-contamination, thelower sealing surfaces and the outlet sized so that the outlet of oneassay unit can seal against the lower sealing surface of another assayunit.
 2. The assembly of claim 1, wherein a pipette tip seals againstthe lower of the two sealing surfaces above the packed bed.
 3. Animmunoassay or selective adsorption assembly comprising a flow-throughassay unit and an aspiration pump; wherein the flow-through assay unitincludes a pair of porous bed supports trapping between them solid phasesupport beads with a reaction reagent mounted on their surface; andwherein the pump includes a piston pump in fluid communication with theflow-through unit to draw fluid therethrough.
 4. An immunoassay orselective adsorption assembly as claimed in claim 3 wherein theflow-through assay unit also includes a pair of sealing surfaces offrusto-conical shape and of differing sizes to accommodate fluid tightcommunication with a variety of fluid handling equipment.
 5. Animmunoassay or selective adsorption assembly as claimed in claim 3wherein the piston pump is vertically oriented with a conical opening atits top, and wherein the assay unit has a corresponding tapered openingat its base so that the assay unit can sit upon the pump in fluid-tightfashion.
 6. A method of performing an analytical assay to identify aanalytical target, the method comprising the steps of: loading anaffinity reagent onto a packed non-porous bed of a flow-through unit ofan assay unit, loading an experimental sample into the packed bed,washing unbound sample out of the packed bed; and eluting the boundsample from the packed bed to identify the target of interest, whereinbefore loading the sample the bed is first loaded with liquid in afashion so as to clear any air out of the packed bed, so that the sampleis loaded in such a manner to minimize air bubbles in the packed bed. 7.The method of claim 6 wherein the loading of the affinity reagent,loading the sample, washing the unbound sample and eluting the boundsample are all driven by an aspirating pump applying negative pressureon the output of the packed bed.
 8. The method of claim 6 wherein theeluted sample is optically sensed to determine the presence of theanalytical target.
 9. A method of performing an analytical assay toidentify a analytical target, the method comprising the steps of:loading an affinity reagent onto a packed non-porous bed of aflow-through unit of an assay unit, loading an experimental sample intothe packed bed, washing unbound sample out of the packed bed;introducing an enzyme reagent into the packed bed which will bind to anybound sample; and passing substrate for the enzyme through the packedbed to identify the target of interest by determining if the substratehas been converted by the enzyme, wherein before loading the sample, thebed is first loaded with liquid in a fashion so as to clear any air outof the packed bed, so that the sample is loaded in a manner so as tominimize air bubbles in the packed bed.
 10. The method of claim 9wherein the loading of the affinity reagent, loading the sample, washingthe unbound sample and passing the enzyme substrate are all driven by anaspirating pump applying negative pressure on the output of the packedbed.
 11. The method of claim 9 wherein the step of introducing theenzyme reagent is performed by introducing an enzyme bound to anantibody to the target.
 12. The method of claim 9 wherein the solutionexiting from the assay unit after passing the substrate is opticallysensed to determine the presence of the analytical target.
 13. A methodof performing an immuno-specific or other selective adsorption assay,the method comprising the steps of (a) providing an assay unit includinga packed bed of beads therein held in place by porous bed supports, thebeads being coated with an immuno-specific or other selective bindingbiological molecule or selective binding surface functionality; (b)introducing into the assay unit an excess of fluid which extends into asample chamber above the packed bed in such a manner to minimize any airentrained in the packed bed; (c) dispensing the test material into thesample chamber; (d) drawing fluid out from the lower porous support todraw fluid containing the test material into the packed bed; (e)permitting the assay to be conducted in the packed bed; and (f) pumpingfluid out of the lower porous support to remove the assay products fromthe packed bed; and (g) detecting the assay products by optical sensing.14. The method as claimed in claim 13 wherein after conducting theassay, an enzyme linked to another immuno-specific biomolecule isintroduced into the packed bed, and the assay product is a product ofconversion of a substrate by the enzyme which only occurs if theenzyme-biomolecule linkage is bound to test material in the packed bed.15. An aspiration pump for use in an assay device, the pump comprising apump body have an inlet port formed in it opening into a cylindricalchamber, the inlet port shaped to seal against an assay unit insertedtherein; a piston in the cylindrical chamber which can be extended orwithdrawn to pump fluid out of the pump or draw fluid into the pump; andthe pump body having a side port opening into a plenum formed in it withthe port opening into the inlet port, the side port connected to fluidsupply and exhaust so that fluid can be supplied to or withdrawn fromthe pump as required, the side port connecting to the inlet port so thatthe point at which the assay unit seals to the inlet port is between theside port and the cylindrical chamber.
 16. An aspiration pump as claimedin claim 15 wherein the cylindrical chamber is oriented vertically andthe inlet port is a conical opening at the top of the cylindricalchamber, the plenum opening into the inlet port.
 17. A packed bed for anassay unit comprising an assay unit body have a cavity in it to receivedthe packed bed; a plurality of non-porous particles having an averagediameter of between 20 and 150 micron located in the packed bed; a pairof porous supports located above and below the particles to confine theparticles in the packed bed; and the bed diameter being between about0.5 and 1.0 mm, the length of the bed between the porous supports beingbetween 1 and 50 mm, so that the dimensions of the bed are optimized forconsistent results.
 18. A packed bed as claimed in claim 17 wherein theparticles have a diameter of between 40 and 100 microns.
 19. A packedbed as claimed in claim 17 wherein the bed diameter is between about 0.7and 0.8 mm.